Regulation of myocardial autophagy by the G protein-coupled estrogen receptor 1 in basal and β adrenergic activated states

Abstract ID 130382 Poster Board 273 Elizabeth McConnaha, Eric Wauson, Jennifer Giles, and Quang-Kim Tran Department of Physiology and Pharmacology, Des Moines University, West Des Moines, IA Objectives: Autophagy is a process that packages and delivers unwanted cellular components to lysosomes for degradation to maintain homeostasis. While myocardial autophagy is cardioprotective, activation of β adrenoceptors (β-ARs) reduce autophagic vacuoles. We previously showed that activation of the G protein-coupled estrogen receptor 1 (GPER, aka GPR30) suppresses the increases in myocardial Ca²⁺, left ventricular contractility, and ectopic contractions triggered by β-AR activation. However, it is unknown whether GPER regulates myocardial autophagy in basal and βAR-activated conditions. Methods: Cardiomyocytes were isolated from WT and GPER−/− C57BL/6 adult male mice. Autophagy was assessed using the autophagic flux assay. Activities of upstream autophagy regulators mTOR and AMPK were assessed by immunoblotting. Starvation-induced Ca²⁺ signal was assessed using AUCs of fractional fura-2 signals that occurred upon amino acid removal. Lysosomal pH was assessed with LysoSensor Yellow/Blue DND160. In heterologous expression paradigms, starvation-induced Ca²⁺ signal and lysosomal pH were compared between WT and GPER-overexpressing cells in the same microscopic fields. Results: Basal autophagic flux is ∼13-fold lower in cardiomyocytes isolated from GPER−/− vs WT male mice, while there are no differences in the activities of mTOR or AMPK. β-AR activation using isoproterenol treatment significantly and dose-dependently reduces autophagic flux in WT cardiomyocytes. In GPER−/− cardiomyocytes, isoproterenol causes further inhibition of autophagic flux, albeit to a lesser degree as in WT cardiomyocytes. Isoproterenol triggers robust intracellular Ca²⁺ signals and significantly reduces lysosomal acidity, an important regulator of autophagic flux. In H9c2 cardiomyocytes, chelation of intracellular Ca²⁺ using the chelator BAPTA-AM significantly increases basal lysosomal pH. BAPTA-AM pretreatment also abolishes the isoproterenol-induced increase in lysosomal pH whereas pretreatment with TF-BAPTA-AM, a 400-fold weaker Ca²⁺ chelator than BAPTA-AM, does not. In GPER−/−primary cardiomyocytes, basal lysosomal acidity is significantly reduced and starvation-induced as well as isoproterenol-stimulated Ca²⁺signals are significantly enhanced. Consistently, GPER overexpression in HEK293 cells promotes lysosomal acidification in basal and starved states and suppresses starvation-induced Ca²⁺ signal. These effects are not affected by truncation of GPER's PDZ-binding motif, a domain responsible for its many interactions with membrane proteins. Conclusions: Our data indicate that 1) β-AR activation suppresses myocardial autophagy via Ca²⁺-dependent alterations in lysosomal pH; 2) expression of GPER plays an important role in controlling basal autophagy by promoting lysosomal acidity; and 3) in stressed states, GPER expression suppresses β-AR-mediated Ca²⁺ signals, reduces starvation-induced Ca²⁺ signal, and enhances starvation-induced lysosomal acidification. Since these effects were observed in cardiomyocytes isolated from male mice and no estrogen or GPER agonist were included, these effects likely reflect estrogen-independent actions of GPER.